ABSTRACT The BRG1 catalytic subunit of SWI/SNF-related complexes is required for mammalian development as exemplified by the early embryonic lethality of Brg1 null homozygous mice. BRG1 is also a tumor suppressor and, in mice, 10% of heterozygous (Brg1(null/+)) females develop mammary tumors. We now demonstrate that BRG1 mRNA and protein are expressed in both the luminal and basal cells of the mammary gland, raising the question of which lineage requires BRG1 to promote mammary homeostasis and prevent oncogenic transformation. To investigate this question, we utilized Wap-Cre to mutate both Brg1 floxed alleles in the luminal cells of the mammary epithelium of pregnant mice where WAP is exclusively expressed within the mammary gland. Interestingly, we found that Brg1(Wap-Cre) conditional homozygotes lactated normally and did not develop mammary tumors even when they were maintained on a Brm-deficient background. However, Brg1(Wap-Cre) mutants did develop ovarian cysts and uterine tumors. Analysis of these latter tissues showed that both, like the mammary gland, contain cells that normally express Brg1 and Wap. Thus, tumor formation in Brg1 mutant mice appears to be confined to particular cell types that require BRG1 and also express Wap. Our results now show that such cells exist both in the ovary and the uterus but not in either the luminal or the basal compartments of the mammary gland. Taken together, these findings indicate that SWI/SNF-related complexes are dispensable in the luminal cells of the mammary gland and therefore argue against the notion that SWI/SNF-related complexes are essential for cell survival. These findings also suggest that the tumor-suppressor activity of BRG1 is restricted to the basal cells of the mammary gland and demonstrate that this function extends to other female reproductive organs, consistent with recent observations of recurrent ARID1A/BAF250a mutations in human ovarian and endometrial tumors.

Full-text

The BRG1 Chromatin Remodeler Protects AgainstOvarian Cysts, Uterine Tumors, and Mammary Tumors ina Lineage-Specific MannerDaniel W. Serber1, Allison Rogala2, Maisam Makarem3,4,5,6, Gary B. Rosson1, Karl Simin1¤a, VirginiaGodfrey2, Terry Van Dyke1¤b, Connie J. Eaves3,4,5,6, Scott J. Bultman1*1Department of Genetics, University of North Carolina, Chapel Hill, North Carolina, United States of America, 2Department of Pathology and Laboratory Medicine,University of North Carolina, Chapel Hill, North Carolina, United States of America, 3Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia,Canada, 4Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada, 5Department of Medicine, University of British Columbia,Vancouver, British Columbia, Canada, 6Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, CanadaAbstractThe BRG1 catalytic subunit of SWI/SNF-related complexes is required for mammalian development as exemplified by theearly embryonic lethality of Brg1 null homozygous mice. BRG1 is also a tumor suppressor and, in mice, 10% of heterozygous(Brg1null/+) females develop mammary tumors. We now demonstrate that BRG1 mRNA and protein are expressed in both theluminal and basal cells of the mammary gland, raising the question of which lineage requires BRG1 to promote mammaryhomeostasis and prevent oncogenic transformation. To investigate this question, we utilized Wap-Cre to mutate both Brg1floxed alleles in the luminal cells of the mammary epithelium of pregnant mice where WAP is exclusively expressed withinthe mammary gland. Interestingly, we found that Brg1Wap-Creconditional homozygotes lactated normally and did notdevelop mammary tumors even when they were maintained on a Brm-deficient background. However, Brg1Wap-Cremutantsdid develop ovarian cysts and uterine tumors. Analysis of these latter tissues showed that both, like the mammary gland,contain cells that normally express Brg1 and Wap. Thus, tumor formation in Brg1 mutant mice appears to be confined toparticular cell types that require BRG1 and also express Wap. Our results now show that such cells exist both in the ovaryand the uterus but not in either the luminal or the basal compartments of the mammary gland. Taken together, thesefindings indicate that SWI/SNF-related complexes are dispensable in the luminal cells of the mammary gland and thereforeargue against the notion that SWI/SNF-related complexes are essential for cell survival. These findings also suggest that thetumor-suppressor activity of BRG1 is restricted to the basal cells of the mammary gland and demonstrate that this functionextends to other female reproductive organs, consistent with recent observations of recurrent ARID1A/BAF250a mutations inhuman ovarian and endometrial tumors.Citation: Serber DW, Rogala A, Makarem M, Rosson GB, Simin K, et al. (2012) The BRG1 Chromatin Remodeler Protects Against Ovarian Cysts, Uterine Tumors, andMammary Tumors in a Lineage-Specific Manner. PLoS ONE 7(2): e31346. doi:10.1371/journal.pone.0031346Editor: Toshi Shioda, Massachusetts General Hospital, United States of AmericaReceived October 12, 2011; Accepted January 6, 2012; Published February 21, 2012Copyright: ? 2012 Serber et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Funding: This work was supported by grants from the National Institutes of Health (National Cancer Institute grant CA125237) and the American Institute forCancer Research to SJB, and the Canadian Breast Cancer Research Alliance (#019343) to CJE. MM is a recipient of a Canadian Institutes of Health Research Bantingand Best Graduate Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing Interests: The authors have declared that no competing interests exist.* E-mail: Scott_Bultman@med.unc.edu¤a Current address: Department of Cancer Biology, University of Massachusetts, Worcester, Massachusetts, United States of America¤b Current address: Mouse Cancer Genetics Program, National Cancer Institute, Frederick, Maryland, United States of AmericaIntroductionMammalian SWI/SNF chromatin-remodeling complexes reg-ulate many cellular processes and function as tumor suppressors.Notably, the BRG1, BRM, SNF5, ARID1A/BAF250a, PBRM1/BAF180, and Srg3/Baf155 subunits are consistently mutated orsilenced in certain primary human tumors and also protectagainst tumorigenesis in mouse models [1–4]. Further evidence ofthe tumor-suppressor role of these genes has come fromexperiments showing that restoration of wild-type expression ofthe mutated or silenced subunit in tumor-derived cell lines candecrease proliferation and promote differentiation [5]. Mecha-nistically, several SWI/SNF subunits have been shown tophysically interact with known tumor-suppressor genes andproto-oncogenes or their encoded proteins [1–3]. These studiesinclude the demonstrated ability of the BRG1 catalytic subunit(also known as SMARCA4) and SNF5 (also known as BRG1-associated factor 47 or BAF47) to bind to the promoters ofthe p15INK4b(known as p19 in the mouse), p16INK4a, andp21CIP1/WAF1cyclin-dependent kinase (CDK) inhibitors andactivate expression of these target genes [6–10]. This, in turn,leads to an inhibition of CDK2 or CDK4 and an accumulation ofhypophosphorylated RB. BRG1 and an alternative catalyticsubunit, BRM (also known as SMARCA2), can also bind tohypophosphorylated RB and are required to repress the activityof E2F1, inhibit the transcription of cyclins A and E, and mediateG1cell-cycle arrest [11–15].We previously showed that the homozygous Brg1 null genotypeis embryonic lethal in mice and 10% of Brg1 null heterozygousmice spontaneously develop mammary tumors at approximatelyPLoS ONE | www.plosone.org1 February 2012 | Volume 7 | Issue 2 | e31346

Page 2

one year of age without prior exposure to ionizing radiation (IR)or other known oncogenic agents [16,17]. These tumors do notshow loss of heterozygosity (LOH) at the Brg1 locus but do exhibitgenomic instability suggesting that the acquisition of secondarymutations in addition to Brg1 haploinsufficiency helps drive thedevelopment of the mammary tumors obtained. Interestingly,these Brg1null/+mammary tumors are more heterogeneous interms of their histopathology, cytokeratin expression, andtranscriptome profiles than the mammary tumors that arise inother mouse models of breast cancer [17]. To further investigatethe role of Brg1 as a tumor suppressor, we now report the resultsof experiments that demonstrate a relationship between thenormal transcriptional activity of the Brg1, Brm and Whey acidicprotein (Wap) genes, and the effect on viability and transformationof Wap-activated deletion of Brg1 in the presence or absence ofBrm.ResultsBrg1 and Brm are co-expressed in all mammary epithelialcells, whereas Wap expression is confined to the luminalcells of the mammary gland in pregnant miceIn a first series of experiments, we sought to characterize theexpression of Brg1, Brm, and Wap in different subsets of mammarycells in normal adult virgin and pregnant female mice.Accordingly, we dissociated their mammary gland fat pads intosingle-cell suspensions, removed hematopoietic, endothelial, andstromal cells, and subdivided the mammary epithelial cells into 3fractions according to their levels of expression of CD24 andCD49f as described [18]. Representative fluorescent activated cellsorter (FACS) plots of the cells isolated for this analysis are shownin Figure 1A for cells from normal virgin mice and in Figure S1 forcells from pregnant mice. We then determined the levels of Brg1,Figure 1. Expression of Brg1, Brm, and Wap in CD452Ter1192CD312mammary gland subpopulations. A. Representative FACS plot ofthe subpopulations remaining after removing the hematopoietic and endothelial (CD45+Ter119+CD31+) cells from a suspension prepared fromdissociated mammary tissue from a normal adult virgin mouse. The CD24+CD49flow/2subset is enriched in luminal (Lum) cells; the CD24+CD49f+subset is enriched in mature myoepithelial (Myo) cells; and the CD24+CD49highsubset is enriched in mammary stem cells (referred to as mammaryrepopulating units or MRU). B, C. RT-qPCR analysis of Brg1 (B) and Brm (C) mRNA levels normalized to Gapdh levels in wild-type, flow-sorted mammarycell populations from adult virgin female mice. The Lum, Myo, and MRU subsets are as defined in panel A. Each histogram represents the mean 6 SEfrom 3 independent experiments. D. RT-qPCR analysis of Wap mRNA levels normalized to Gapdh mRNA levels measured in the same 3 subsets. Lum,Myo, and MRU cells were isolated from pregnant females (E17.5 and E13.5) and virgin/nulliparous females (for representative FACS plots, see Figure 1).Asterisks indicate that the Wap signal was below the limit of detection. Each histogram represents the mean 6 SE from 2 (E17.5) or 3 (E13.5, virgin/nulliparous) independent experiments.doi:10.1371/journal.pone.0031346.g001BRG1 Mammary and Female Reproductive Tract TumorsPLoS ONE | www.plosone.org2 February 2012 | Volume 7 | Issue 2 | e31346

pregnancy (E17.5) but not at an earlier stage (E13.5), nor invirgin/nulliparous females (Figure 1D). This is consistent with theknown period of hormonally-induced Wap expression [20]. Waptranscripts were also not detectable in either of the other 2 subsetsof basal mammary epithelial cells (Figure 1D). These findingspredicted that a Wap-Cre transgene would delete Brg1 only in theluminal cells of the mammary gland of mice and not until the micehad reached a late stage of pregnancy.Luminal mammary cells lacking Brg1 remain viable andfully functional and do not generate tumorsTo evaluate the function of BRG1 in the luminal lineage, wegenerated 21 female mice carrying a floxed Brg1 gene and a Wap-Cre transgene [21,22] (either as Brg1fl/fl:Wap-Cre+/0mice or asBrg1null/fl:Wap-Cre+/0mice, hereafter referred to as Brg1Wap-Cremutants), and then monitored them for 15–19 months withoutexposure to IR or any other known oncogenic agents. To deleteBrg1 in the luminal cells, we mated all 21 females several times,starting at 2–3 months of age. Twelve littermates were monitoredin parallel as controls. All of the females produced at least 2 litterseach, and none showed any evidence of subsequent abnormalities.In particular, none of the mice developed any signs of mammarytumor formation (Table 1). Whole-mount preparations of theirmammary glands removed after 15–19 months also did not revealany evidence of microscopic tumors or altered morphology of theirmammary glands (Figure 2A).PCR assays of cells harvested from the mice at the end of theBrg1Dflallele monitoring period confirmed that the Brg1flallele hadbeen converted to a recombined in mammary glands frommultiparous but not nulliparous females (Figure 2B). Detection ofthe fl allele in the PCR assays was not unexpected becausemammary epithelial cells are embedded in a fat pad that alsocontains lymph nodes and blood vessels, and Wap-Cre is notexpressed in adipocytes, lymphocytes, or other stromal cells suchas fibroblasts. The Brg1DflPCR product also could not be detectedin tail tissue from either multiparous or nulliparous females(Figure 2B) consistent with the target specificity of action of theWap-Cre transgene [22–24].To further characterize the mammary cells in which Brg1 isdeleted in Brg1Wap-Cremutants, we introduced the Rosa26 reporter(R26R) gene into the Brg1Wap-Cremice [25]. X-Gal staining of tissueremoved from multiparous females that were not at that timeeither pregnant, lactating, or involuting confirmed that the Wap-activated Cre activity had been restricted to the luminal cells of theFigure 2. Lineage-specific deletion of Brg1 in the mammary gland. A. Whole-mount preparation of control (top) and mutant (bottom)mammary glands from multiparous females. Asterisks, lymph nodes. B. Ethidium bromide-stained gels showing Brg1 Dfl (top) and fl (bottom) PCRproducts. MW, molecular-weight standard (500-, 400-, 300-, 200-, and 75-bp fragments are visible); MG, mammary gland; NP, nulliparous; MP,multiparous. C. Mammary gland section from multiparous mouse carrying the Wap-Cre transgene on a R26R background. Cre activity, visualized asblue X-Gal staining, is restricted to luminal cells (arrowhead). Basal/myoepithelial cells (arrow) are negative and appear pink because of nuclear fastred counterstain. D, E. IHC staining of BRG1 showing strong staining in nuclei throughout the mammary gland in controls (D) but absent in luminalcells of mutant mice (E). Arrows, luminal cells; asterisks, adipocyte nuclei. 4006magnification.doi:10.1371/journal.pone.0031346.g002BRG1 Mammary and Female Reproductive Tract TumorsPLoS ONE | www.plosone.org4 February 2012 | Volume 7 | Issue 2 | e31346

Page 5

mammary epithelium (Figure 2C). Next, we performed immuno-histochemical (IHC) studies of sections of mammary tissue frommultiparous Brg1Wap-Cremutants and control nulliparous mice.These experiments showed that the usually strong and widespreadnuclear staining for BRG1 protein (Figure 2D and Figure S2A)was diminished or absent in the luminal cells of the conditionalmutants while persisting in stromal cells of the same animals whereWap-Cre is not expressed and Brg1 was not mutated (Figure 2E).Taken together, these results demonstrate that Brg1 is mutated inthe expected luminal lineage cell-specific manner and arguesstrongly against a defect in Cre-mediated recombination as anexplanation for the lack of mammary tumors in Brg1Wap-Cremutantmice.Mammary luminal cells lacking Brg1 do not undergoaberrant apoptosisIt was reported that a heterozygous deletion of Brg1 in certainlung cells, makes them prone to urethane-induced tumorigenesis,whereas a homozygous deletion of Brg1 in the same cells does notbecause the homozygous deletion induces apoptosis [26]. Toinvestigate whether aberrant apoptosis (unrelated to involution)might account for the lack of mammary tumors in Brg1Wap-Cremutant mice, we performed TUNEL assays on mammary glandsharvested from multiparous females that were not pregnant,lactating, or involuting at the time. In controls, we observedminimal apoptosis as only 0.25% of the mammary epithelial cellswere TUNEL-positive (Figure 3A), which is consistent withprevious reports of wild-type mammary glands [27]. And incontrast to the lung tumor study, we did not detect a significantdifference in the frequency of apoptosis in mutants, where only0.20% of the cells were TUNEL positive (Figure 3B). In theseexperiments, a leukemia cell line induced to undergo apoptosisserved as a positive control for the TUNEL staining (Figure 3C).We also performed qPCR analyses to determine whether Brg1-deleted cells are significantly underrepresented when the abun-dance of the Dfl PCR product in mammary gland tissue fromconditional homozygous (Brg1fl/fl:Wap-Cre+/0) and conditionalheterozygous (Brg1fl/+:Wap-Cre+/0) multiparous females was com-pared. Unlike the lung cancer study, which reported a 1:8 ratio ofDfl PCR product in homozygous versus heterozygous mice [26],we obtained a ,2:1 ratio of Dfl PCR product in a comparison ofour homozygous and heterozygous mice, which was what wasexpected if the Dfl/Dfl cells were not being strongly selectedagainst by activation of either apoptosis or necrosis mechanisms(Figure 3D).Finally, we noted that every Brg1Wap-Cremutant female thatbecame pregnant not only delivered a healthy litter but was alsoable to lactate and raise normal-sized pups. This finding indicatesthat the alveolar cell proliferation and differentiation that underliesmammary gland development during pregnancy and lactation hadnot been impaired in spite of the lack of Brg1 expression. Asignificant increase in apoptosis would have resulted in insufficientFigure 3. Mutant luminal cells do not undergo aberrant apoptosis. A, B. TUNEL assay of control (A) and mutant (B) adult mammary glandsections counterstained with methyl green. C. Lymphocytes induced to undergo apoptosis have brown nuclei (arrow) and serve as a positive control.D. qPCR analysis of Brg1 Dfl genomic DNA levels normalized to Gapdh genomic DNA levels in mammary glands. Each histogram represents the mean6 SE from 3 independent experiments.doi:10.1371/journal.pone.0031346.g003BRG1 Mammary and Female Reproductive Tract TumorsPLoS ONE | www.plosone.org5 February 2012 | Volume 7 | Issue 2 | e31346

Page 6

milk production and neonatal runting or lethality of their progenyas observed for mammary-specific mutations of other genes [28–33]. Thus, overall we could not find any evidence of decreasedviability of Brg1-deficient luminal cells.Brm is not required to compensate for the loss of Brg1 inluminal mammary cellsBRG1 and BRM are 75% identical, broadly expressed, andserve as alternative catalytic subunits of SWI/SNF-relatedcomplexes with similar or identical activities [34–35]. Todetermine whether the lack of a phenotype in Brg1-deletedluminal mammary cells might be explained by functionalcompensation from the co-expressed Brm, we transferred theBrg1Wap-Cremutation onto a Brm-deficient background. We thenmonitored 8 double-mutant females and 6 control (non-transgenic)females for 15–19 months without exposure of either to IR or anyother known oncogenic agents (Table 2). To inactivate Brg1 ineach of the 14 females, a first pregnancy was initiated at 2–3months of age, and then again 1–2 times subsequently. From eachof these mice, 2–3 litters of normal sized pups were obtained,indicating that the luminal cells of the mammary gland can remainviable and completely functional even when neither Brg1 nor Brmis present. Nor did any of these mice develop mammary tumors(Table 2). These results demonstrate that the ‘‘normal’’ phenotypeof Brg1 null luminal cells is not due to a compensatory activitybeing provided by Brm nor are either of these catalytic subunitsrequired to prevent these cells from generating tumors.Mammary tumor induction caused by inhibition of theRB pathway is not altered by Wap-Cre-mediated deletionof Brg1 in luminal cellsBRG1 interacts with RB and is required for RB-mediatedgrowth arrest in tumor-derived cell lines in vitro [11–15]. However,it is not clear whether this mechanism applies to cancer preventionin vivo. In a previous study, we noted that the induction of Rb+/2mammary tumors was not altered on a Brg1null/+background [17].To determine whether this would also extend to Brg1 null luminalcells, we crossed Wap-T121 transgenic mice with our Brg1Wap-Cremutants and then monitored them for tumor formation. T121contains the first 121 amino acids of the SV40 large T antigen,which binds to RB as well as the other 2 pocket proteins (p107 andp130) and perturbs their function [32]. Wap-T121 mice expressthis transgene in their mammary luminal cells and developaggressive mammary tumors with 100% penetrance by ,16months of age [32]. We confirmed this finding but observed nofurther change in the penetrance or latency (Figure S3) or thehistopathologic characteristics (data not shown) of the tumorphenotype in 10 Wap-T121, Brg1Wap-Credouble mutant mice ascompared to mice expressing only Wap-T121.Wap-Cre-mediated deletion of Brg1 induces ovarian cystsand uterine tumor formationAlthough Brg1Wap-Cremultiparous females are not predisposed todevelop breast cancer, we discovered that they do develop grosslyvisible ovarian cysts and uterine neoplasms in contrast to age-matched sibling control females in none of whom such pathologiesoccurred (Table 1). The ovarian cyst phenotype was highly (76%)penetrant, affecting 16 out of 21 conditional-mutant females. Thecystic ovaries ranged in size from 5–12 mm (Figure 4A) and wereunilateral in 12/16 females and bilateral in 4/16 females. Theaffected ovaries contained 1–3 cysts, which is less than the numberpresent in polycystic ovaries [36]. In addition, Brg1Wap-Cremutantfemales did not exhibit hirsutism or reduced fertility as youngadults over several litters, which is characteristic of humanpolycystic ovarian disease caused by altered hormonal influencessuch as hyperandrogenism [37]. We also did not detect asignificant difference in circulating progesterone levels (FigureS4), which might have provided insight into the polycystic ovariesor the lack of mammary tumors in Brg1Wap-Cremutants.Histopathological analysis of H&E-stained ovary sections revealedfollicular cysts lined with either spindle cells or columnar cellscharacteristic of having apical ciliary structures (Figure 4B). All cystscontained either serous fluid or varying stages of hemorrhageTable 2. Phenotype of Brg1Wap-Cremutant mice on a Brm-deficient background.PhenotypeGenderCategoryGenotype1#Litters* MammaryOvary/UterusFemale control floxed/floxed, Brm2/23nonenoneFemale controlfloxed/floxed, Brm2/3nonenoneFemale controlfloxed/floxed, Brm2/23nonenoneFemale controlfloxed/floxed, Brm2/2floxed/floxed, Brm+/22nonenoneFemale control2none noneFemale controlfloxed/floxed, Brm2/23 none noneFemaledouble mutantfloxed/floxed, Tg, Brm2/23 none noneFemaledouble mutantfloxed/floxed, Tg, Brm2/23noneovarian cystFemaledouble mutant floxed/floxed, Tg, Brm2/22none ovarian cystFemaledouble mutant floxed/floxed, Tg, Brm2/22none noneFemaledouble mutantfloxed/floxed, Tg, Brm2/23noneovarian cystFemaledouble mutantfloxed/floxed, Tg, Brm2/22noneovarian cystFemaledouble mutant floxed/floxed, Tg, Brm2/23nonenoneFemaledouble mutant floxed/floxed, Tg, Brm2/22noneovarian cyst*First pregnancy was at 2–3 months of age, no lactation defects were observed, and all mice were analyzed at 15–19 months of age.1Tg was hemizygous in each case (Wap-Cre+/0).doi:10.1371/journal.pone.0031346.t002BRG1 Mammary and Female Reproductive Tract TumorsPLoS ONE | www.plosone.org6 February 2012 | Volume 7 | Issue 2 | e31346

Page 7

ranging from acute to chronic basedupon the degree of organization(Figure 4B, C). Serous cysts were encapsulated by an inner layer ofspindle-shaped cells, a middle layer of granulosa cells, and an outerlayer of spindle cells (Figure 4C). The uterine neoplasm phenotypewas 19% penetrant, occurring in 4 of 21 conditional mutant females(Table 1). These neoplasms were of 2 histopathologic types: eitherhistiocytic sarcomas projecting into the uterine lumen (Figure 4D) orendometrial stromal polyps (Figure 4E, F).IHC analysis demonstrated that BRG1 is normally expressed inboth the ovary and the uterus. In the ovary, BRG1 protein wasdetected in oocytes, granulosa cells, and theca cells within thefollicles (Figure S2B). In the uterus, BRG1 was expressed in awidespread manner (Figure S2C). In the experiments where weperformed X-Gal staining on R26R:Wap-Cre+/0tissues, Creactivity was detected consistently in the ovary and uterus as wellas in the mammary glands of 10 Wap-Cre transgenic mice but notin negative controls carrying R26R but lacking the Wap-Cretransgene. These findings are consistent with endogenous Wapexpression in the ovary and uterus [37]. Ovarian Cre activity wasobserved in granuolosa cells within follicles (Figure S5). Similarly,BRG1 immunostaining was abolished or diminished in these cellsin the Brg1Wap-Cremutants (Figure S2D, E). The results presentedabove strongly suggest that the generation of the ovarian cysts anduterine tumors is a cell autonomous process.Figure 4. Histopathology of ovarian cysts and uterine tumors from Brg1Wap-Cremice. A. Control ovary (left) and cystic ovary from aconditional Brg1Wap-Cremutant (right). B. Section of an ovarian cyst stained with H&E at 406magnification. The thin wall of this multilocular structureis comprised of an inner layer of spindle-shaped cells surrounded by multiple layers of round cells with uniformly sized, round, basophilic nuclei withscant cytoplasm (arrows). This cyst is filled with a lightly eosinophilic proteinaceous fluid (arrowheads). C. Hemorrhagic ovarian cyst section stainedwith H&E at 406magnification. The thin arrow points to a wall of the cyst, and the arrowhead points to a region of hemosiderin-laden macrophagesat the edge of the hemorrhage. D. Histiocytic sarcoma section stained with H&E at 206 magnification. A pleomorphic population of cells withbasophilic round to ovoid nuclei and cytoplasm varying from scant to abundant and foamy are seen infiltrating the myometrium (thin arrows) andforming a polyp (arrowheads) projecting into the uterine lumen E. Endometrial stromal polyp section stained with H&E at 206magnification. Thepolyp (arrows) includes a pedunculated mass (arrowheads) that projects into the uterine lumen. F. Endometrial stromal polyp section stained withH&E stained at 2006 magnification. The polyp stroma is comprised of spindle cells with variable amounts of cytoplasm and ovoid nuclei; thestructure also includes small blood vessels and small endometrial glands and is covered by a single layered cuboidal to columnar epithelium withbasally located round nuclei.doi:10.1371/journal.pone.0031346.g004BRG1 Mammary and Female Reproductive Tract TumorsPLoS ONE | www.plosone.org7 February 2012 | Volume 7 | Issue 2 | e31346

Page 8

DiscussionWe previously demonstrated that Brg1null/+mice are susceptibleto mammary tumorigenesis, but the cells of origin were notidentified [16,17]. We now show that this is unlikely to be aconsequence of decreased Brg1 expression in luminal cells asmammary tumors were never obtained in Brg1Wap-Creconditionalmutants in spite of the fact that Brg1 is normally expressed in thesecells and was successfully deleted by forcing the mice to undergomultiple pregnancies but without any evidence of increasedapoptosis. These findings imply that Brg1 haploisufficiency mustactivate an oncogenic process in other cells, either members of thebasal mammary cell compartment and/or stromal cells. Consistentwith this hypothesis is our finding that Brg1 is normally expressed inthebasalcells ofthemammarygland buttheBrg1 floxed allele couldnot be deleted in Brg1Wap-Creconditional mutant mice becauseexpression of Wap and hence Cre is not induced in the basal cells.Additional support for an important role of Brg1 in mammary stemcellsis the demonstration ofitsrequirementforembryonicstem (ES)cell self-renewal and pluripotency [38,39], as well as other, morerestricted, types of stem cells [40,41]. A Brg1 stem/progenitor cell-restricted function in the mammary gland is also consistent with thediverse histopathological characteristics and transcriptome profilesofBrg1null/+tumors[17].ItmayalsoexplainwhydepletionofBRG1from MCF-10A mammary cells, which have characteristics of non-malignant luminal cells, did not increase their proliferative activitynor confer a tumor-like phenotype [42].The restricted expression of Wap-Cre to the luminal cells in themammary gland may explain why only certain Wap-Creconditional mutants develop highly penetrant tumor phenotypes.For example, Wap-Cre driven mutation of Smad4 and Brca2 causesa very high frequency of affected mice to develop mammarytumors (100% and 77%, respectively), whereas only 15% of micewith Wap-Cre driven mutation of Brca1 develop mammary tumorsand no tumors are obtained following Wap-Cre driven mutation ofPparc or Stat3 [43–47]. Failure of tumorigenesis would be expectedif the gene being targeted for deletion was not expressed in aluminal cell, or not required, or involved in a pathway whoseperturbation would lead to deregulated growth of a luminal cell. Inaddition, Wap-Cre driven mutations that require other genetic orepigenetic changes to be accumulated might not lead to tumorsbecause of the transient lifespan of the luminal compartment.SWI/SNF-related complexes are essential for the developmentof many cell lineages [48], which suggests that they might berequired for the viability of most or all primary cell types. In fact,the only cells previously known to deficient for both BRG1 andBRM are certain tumor-derived cell lines [2,5], and these tumorcells may have subverted the normal requirement for at least onecatalytic subunit via inhibition of apoptosis. However, our presentfindings now offer a potential alternative explanation; i.e., thatSWI/SNF-related complexes may be dispensable in mammaryluminal cells. Here we show that neither Brg1 nor Brm are requiredfor a morphologically- and functionally-normal mammary glandand their absence did not affect the ability of the gland to supportthe repeated production of litters of normal-sized pups. Thus,SWI/SNF-related complexes catalyzed by either BRG1 or BRMmust be dispensable for the viability and normal functionality ofmammary luminal cells, in spite of previous experiments withEpH4 cells expressing a dominant-negative BRG1 suggesting thatcasein expression is dependent on SWI/SNF catalytic activity[49]. It is likely that other primary cell types can also survive in theabsence of BRG1/BRM-catalyzed SWI/SNF complexes, and wehave evidence from Villin-Cre experiments that this is the case forintestinal epithelial cells (data not shown).Although Brg1Wap-Cremice did not develop mammary tumors,they did become susceptible to the formation of ovarian cysts anduterine tumors. The ovarian cysts were associated with a loss ofBRG1 in granulosa cells (Figure S5), whereas our previous analysisof Brg1Zp3-Creconditional mutants indicated that ovarian cysts didnot develop when Brg1 was deleted in developing oocytes [50].These results are compatible with ovarian cysts arising fromfunctional defects in somatic support cells rather than germ cells.BRG1 could prevent cyst formation in wild-type cells through itswell-known role in development and differentiation [2,48].Alternatively, it could be promoting apoptosis to promote thedeath of immature ovarian follicles (i.e., artesia) [36]. The uterineneoplasms are also noteworthy because few genetically engineeredmouse models of uterine cancer other than Pten have beendescribed [51–53]. The Brg1Wap-Creovarian cyst and uterine tumorphenotype is also reminiscent of recent deep-sequencing effortsdemonstrating consistent mutations of another SWI/SNF subunit,ARID1A/BAF250a, in ,30% and ,50% of human ovarian clearcell carcinomas and endometrial carcinomas, respectively [54,55].Our data also support the observation that BRG1 is downregu-lated in human cervical carcinomas [56]. In summary, our resultsadd weight to the idea that SWI/SNF-related complexes have animportant function in preventing the development of cancers,particularly within the stem/progenitor compartments of cells incertain tissues.Materials and MethodsEthics statement, mice, and genotypingAll mouse experiments were approved by the InstitutionalAnimal Care and Use Committees (IACUC) review board at theUniversity of North Carolina as approved protocol ID #10-026and were performed in accordance with federal guidelines. Wap-Cre transgenic mice were obtained from the Jackson Laboratory(Bar Harbor, ME, USA). The Brg1 floxed and Dfloxed alleles weregenotyped by PCR as previously described [21]. Quantification ofthe relative abundance of the Brg1 Dfloxed allele was performed byqPCR and normalized to Gapdh as previously described [26].HistologyNormal tissues and cystic or tumor tissues were fixed in 4%paraformaldehyde, embedded in paraffin, and 5 mm sections werecut according to standard procedures. Sections were either stainedwith H&E or processed for IHC using a BRG1 rabbit polyclonalantibody (Upstate/Millipore #07-478, Temecula, CA, USA) orfor TUNEL assays (Chemicon/Millipore, Temecula, CA, USA)according to the manufacturer’s recommendations. The TUNELpositive control was provided by the manufacture and consisted ofhuman promyelocytic leukemia treated with actinomycin D.Whole mount preparations of mammary glands and X-Galstaining of R26R (Rosa-lox-stop-lox-LacZ) tissues were performedfollowing standard procedures. Serum progesterone levels weredetermined by ELISA.Isolation of mammary cell populationsMammary glands were dissected from 8–12 week old, femaleC57BL/6J mice before or after induction of pregnancy (E13.5 orE17.5) as indicated. Single-cell suspensions were generated andanalyzed by flow cytometry as previously described with minormodifications [18]. Briefly, mammary glands were digestedovernight at 37uC in DMEM/F12 medium containing 1 mg/mL collagenase A (Roche) and 100 U/mL hyaluronidase (Sigma).After vortexing and lysis of red blood cells in NH4Cl, the nucleatedcells were further dissociated in 0.25% trypsin, 5 mg/mL dispaseBRG1 Mammary and Female Reproductive Tract TumorsPLoS ONE | www.plosone.org8February 2012 | Volume 7 | Issue 2 | e31346

Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed.
The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual
current impact factor.
Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence
agreement may be applicable.

[Show abstract][Hide abstract]ABSTRACT:
Transcription factors (TFs) and microRNAs (miRNAs) can jointly regulate transcriptional network in the forms of recurrent circuits or motifs. A motif can be divided into feedforward loop (FFL) and feedback loop (FBL). Incoherent FFLs have been the recent focus due to their potentials to dampen gene expression noise in maintaining physiological norms. However, cell is not only able to manage noise but also able to exploit it during development or tumorigenesis to initiate radical transformation such as cell differentiation or metastasis. A plausible mechanism may involve reinforcing FBLs (rFBLs), which amplifies changes to a sufficient level in order to complete the state transition. To study the behaviour of rFBL, we developed a novel theoretical framework based on biochemical kinetics. The proposed rFBL follows a parsimonious design, involving two TFs and two miRNAs. Simulation study based on our model suggested that a system with rFBL is robust to only certain level of fluctuation but prone to a complete paradigm shift when the change exceeds a threshold level. To investigate the natural occurrence of rFBL, we performed a rigorous network motif analysis using recently available TF/miRNA regulatory network from Encyclopedia of DNA Elements (ENCODE). Our analysis suggested that the rFBL is significantly depleted in the observed network. Nonetheless, we identified 9 rFBL instances. Among them, we found a double-rFBL involving three TFs SUZ12/BCLAF1/ZBTB33 and three miRNAs miR-9/19a/129-5p, which together serve as an intriguing toggle switch between nerve development and telomere maintenance. Additionally, we interrogated the interactions implicated in the rFBLs using expression profiles of cancer patients from The Cancer Genome Atlas (TCGA). Together, we provided a novel and comprehensive view of the profound impacts of rFBL and highlighted several TFs and miRNAs as the leverage points for potential therapeutic targets in cancers due to their eminent roles in the identified rFBLs.

[Show abstract][Hide abstract]ABSTRACT:
SWI/SNF complexes utilize BRG1 (also known as SMARCA4) or BRM (also known as SMARCA2) as alternative catalytic subunits with ATPase activity to remodel chromatin. These chromatin-remodeling complexes are required for mammalian development and are mutated in ~20% of all human primary tumors. Yet our knowledge of their tumor-suppressor mechanism is limited. To investigate the role of SWI/SNF complexes in the DNA-damage response (DDR), we used shRNAs to deplete BRG1 and BRM and then exposed these cells to a panel of 6 genotoxic agents. Compared to controls, the shRNA knockdown cells were hypersensitive to certain genotoxic agents that cause double-strand breaks (DSBs) associated with stalled/collapsed replication forks but not to ionizing radiation-induced DSBs that arise independently of DNA replication. These findings were supported by our analysis of DDR kinases, which demonstrated a more prominent role for SWI/SNF in the activation of the ATR-Chk1 pathway than the ATM-Chk2 pathway. Surprisingly, γH2AX induction was attenuated in shRNA knockdown cells exposed to a topoisomerase II inhibitor (etoposide) but not to other genotoxic agents including IR. However, this finding is compatible with recent studies linking SWI/SNF with TOP2A and TOP2BP1. Depletion of BRG1 and BRM did not result in genomic instability in a tumor-derived cell line but did result in nucleoplasmic bridges in normal human fibroblasts. Taken together, these results suggest that SWI/SNF tumor-suppressor activity involves a role in the DDR to attenuate replicative stress and genomic instability. These results may also help to inform the selection of chemotherapeutics for tumors deficient for SWI/SNF function.